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Faculty papers Farber Institute for Neurosciences Farber Institute for Neurosciences
1-1-2013
Impairments in neurogenesis are not tightly linkedto depressive behavior in a transgenic mouse modelof Alzheimer's disease.Daniel M IasconeThomas Jefferson University
Sneha PadidamThomas Jefferson University
Mark S PyferThomas Jefferson University
Xiaohong ZhangThomas Jefferson University
Lijuan ZhaoThomas Jefferson University
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Recommended CitationIascone, Daniel M; Padidam, Sneha; Pyfer, Mark S; Zhang, Xiaohong; Zhao, Lijuan; and Chin,Jeannie, "Impairments in neurogenesis are not tightly linked to depressive behavior in a transgenicmouse model of Alzheimer's disease." (2013). Faculty papers Farber Institute for Neurosciences. Paper22.http://jdc.jefferson.edu/farberneursofp/22
AuthorsDaniel M Iascone, Sneha Padidam, Mark S Pyfer, Xiaohong Zhang, Lijuan Zhao, and Jeannie Chin
This article is available at Jefferson Digital Commons: http://jdc.jefferson.edu/farberneursofp/22
Impairments in Neurogenesis Are Not Tightly Linked toDepressive Behavior in a Transgenic Mouse Model ofAlzheimer’s DiseaseDaniel M. Iascone, Sneha Padidam, Mark S. Pyfer, Xiaohong Zhang, Lijuan Zhao, Jeannie Chin*
Department of Neuroscience and Farber Institute for Neurosciences, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America
Abstract
Alzheimer’s disease (AD), the most common cause of dementia, is also associated with depression. Although the precisemechanisms that lead to depression in AD are unknown, the impairments in adult hippocampal neurogenesis observed inAD may play a role. Adult-born neurons play a critical role in regulating both cognition and mood, and reducedhippocampal neurogenesis is associated with depression in other neurological disorders. To assess the relationship betweenAlzheimer’s disease, neurogenesis, and depression, we studied human amyloid precursor protein (hAPP) transgenic mice, awell-characterized model of AD. We report that reductions in hippocampal neurogenesis are evident early in diseaseprogression in hAPP mice, but a mild depressive phenotype manifests only in later stages of disease. We found that hAPPmice exhibited a reduction in BrdU-positive cells in the subgranular zone of the dentate gyrus in the hippocampus, as wellas a reduction in doublecortin-expressing cells, relative to nontransgenic controls at 5–7 months of age. These alterations inneurogenesis appeared to worsen with age, as the magnitude of reduction in doublecortin-expressing cells was greater inhAPP mice at 13–15 months of age. Only 13–15 month old hAPP mice exhibited depressive behavior in the tail suspensiontest. However, mice at both age groups exhibited deficits in spatial memory, which was observed in the Morris water mazetest for hippocampus-dependent memory. These findings indicate that neurogenesis impairments are accompanied bycognitive deficits, but are not tightly linked to depressive behavior in hAPP mice.
Citation: Iascone DM, Padidam S, Pyfer MS, Zhang X, Zhao L, et al. (2013) Impairments in Neurogenesis Are Not Tightly Linked to Depressive Behavior in aTransgenic Mouse Model of Alzheimer’s Disease. PLoS ONE 8(11): e79651. doi:10.1371/journal.pone.0079651
Editor: Javier Vitorica, Universidad de Sevilla, Spain
Received March 20, 2013; Accepted October 4, 2013; Published November 14, 2013
Copyright: � 2013 Iascone et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The Chin lab is grateful for support from the Alzheimer’s Association grant NIRG-11-205769 (www.alz.org) and the Margaret Q. Landenberger ResearchFoundation (no public URL), from whom funds were used to partially support lab staff and animal maintenance during the course of this study. The funders hadno role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interest exist.
* E-mail: Jeannie.Chin@jefferson.edu
Introduction
Alzheimer’s disease (AD) is the most common form of dementia
and is characterized by prominent impairments in memory [1].
The amyloid precursor protein and the amyloid-b (Ab) peptides
cleaved from it play central roles in AD [2,3]. Ab is prone to
aggregation, and deposits into extracellular plaques that are
pathological hallmarks of AD. However, smaller Ab oligomers
disrupt neuronal function early in disease progression, before
plaques begin to form in mouse models of AD [3,4]. The precise
mechanisms through which Ab impairs neuronal function are
unclear.
Neurogenesis is also altered early in disease progression, prior to
plaque deposition in AD mouse models, suggesting that it could
play a role in early cognitive and/or psychiatric symptoms of AD
[5–8]. In adult organisms, neurogenesis occurs in only two specific
brain regions, one of which is the subgranular zone of the
hippocampus [9]. The hippocampus is critical for learning and
memory as well as mood regulation, and adult neurogenesis is
necessary for normal function [9–11]. Notably, the hippocampus
is also particularly vulnerable in AD [12,13]. A number of reports
have demonstrated that adult neurogenesis is altered in AD
patients as well as mouse models; both increases and decreases in
neurogenesis have been reported which may reflect different stages
of disease [5–7,14].
Several studies have examined the implications of impaired
neurogenesis for cognitive deficits in AD, but few have examined
whether neurogenesis impairments are related to the increased
incidence of depression observed in AD [7,15]. Experimentally
reduced neurogenesis alone is not sufficient to induce depressive
behavior in rodents, although rodent models of depression are
associated with deficits in adult neurogenesis [16,17]. Moreover,
the therapeutic effects of certain classes of antidepressants rely
upon mechanisms that increase neurogenesis [16–18]. Thus, there
is a complex relationship between alterations in neurogenesis and
depression [19]. Because neurogenesis regulates critical functions
in the adult brain, and is affected early in AD [7,10,11,14], it is
important to understand the consequences of impaired neurogen-
esis to AD pathophysiology.
We investigated the relationship between impairments in
neurogenesis and development of depressive behavior in trans-
genic mice that overexpress human amyloid precursor protein
carrying AD-associated mutations (hAPP mice), a well-character-
ized model of AD. We assessed depressive behavior using the tail
suspension test, and analyzed hippocampal neurogenesis using
BrdU to label dividing cells as well as immunophenotyping to
identify and count doublecortin-expressing cells. We examined
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hAPP mice at two ages, and found that aged hAPP transgenic
mice exhibit both impaired neurogenesis and depressive behavior,
but that deficits in neurogenesis develop well before depressive
behavior is observed. Thus, impairments in hippocampal neuro-
genesis are not tightly linked to depressive behavior in hAPP
transgenic mice.
Materials and Methods
Ethics StatementAll animal work was performed with the approval of the
Thomas Jefferson University Institutional Animal Care and Use
Committee, under protocol #884A.
Transgenic MicehAPP transgenic line J20 heterozygously expresses hAPP
carrying the Swedish (K670N, M671L) and Indiana (V717F)
familial AD (FAD) mutations (hAPP770 numbering). Transgene
expression in this line is directed by the platelet-derived growth
factor b chain promoter (Mucke et al., 2000). This line has been
crossed for .10 generations onto a C57BL/6 background using
nontransgenic (NTG) C57BL/6 mice from The Jackson Labora-
tory (Bar Harbor, ME). hAPP mice are bred, maintained, and
analyzed as heterozygous transgenic mice, and age-matched NTG
littermates from the same line are used as controls. Before
behavioral testing, mice were singly housed and allowed to
acclimate for at least 4 days prior to testing. Age-matched hAPP
and NTG mice were evaluated at 5–7 months of age (n = 12/
genotype for doublecortin expression studies, n = 6–7/genotype
for BrdU experiments, n = 12/genotype for water maze studies,
n = 24/genotype for depression studies) and 13–15 months of age
(n = 11–12/genotype for doublecortin expression, BrdU, and
depression studies, n = 7–11 for water maze studies). Cohorts of
mixed sex (containing equivalent numbers of male and female
mice) were used for depression and neurogenesis experiments, no
sex differences were found. Two separate cohorts of male mice
were used for water maze experiments. All behavioral experiments
took place during the light phase. At least three days after
behavioral studies were complete, mice were anesthetized and
flush-perfused transcardially with PBS. Brains were fixed in 4%
phosphate-buffered paraformaldehyde.
BrdU labelingBrdU (150 mg/kg, Sigma, St. Louis, MO) was dissolved in
saline and administered intraperitoneally twice, spaced 2 hours
apart. Mice were sacrificed two hours after the last BrdU injection.
Brain sections were stained with rat-anti-BrdU antibody (Accu-
rate, Westbury, NY) followed by donkey-anti-rat secondary
antibody conjugated to AlexaFluor-594 (Invitrogen, Grand Island,
NY). Prolong Gold antifade mounting medium with DAPI
(Invitrogen) was used to allow visualization of nuclei. Labeling
was visualized with epifluorescence microscopy. BrdU-positive
cells in the subgranular zone of the hippocampus were counted in
every 10th coronal section throughout the dorsal-ventral extent of
the hippocampus, and summed.
ImmunohistochemistrySliding microtome sections (30 mm) were avidin-biotin/immu-
noperoxidase stained using an anti-doublecortin antibody (Cell
Signaling, Danvers, MA) followed by biotinylated goat anti-rabbit
antibody (Vector Laboratories, Burlingame, CA). Diaminobenzi-
dine was used as the chromagen. Doublecortin-immunoreactive
neurons in the subgranular zone of the hippocampus were counted
in every 10th coronal section throughout the dorsal-ventral extent
of the hippocampus, and summed. Neuroblasts were distinguished
from immature neurons on the basis of morphology [20,21].
Tail Suspension TestMice were suspended 53 cm above a cushioned pad, using tape
to attach their tails to a horizontal pole above the pad (protocol
adapted from [22]. A tail restriction device was used to prevent the
animals from climbing their tails. Each mouse was tested for a
6 minute trial at baseline and 24 hours later. Latency to the first
bout of immobility (defined as $5 second-long segment of time
spent immobile) and the total time spent immobile during the trial
were recorded by two independent experimenters blinded to
genotype. Immobility was defined as hanging passively without
any movement of the head or paws.
Morris Water MazeMale hAPP and NTG mice were housed in the testing room at
least 4 days prior to testing in the water maze based on our
previously published protocol [23,24]. Mice were tested in a white
circular pool (1.2 m diameter) filled with 21–22uC water made
opaque with nontoxic white paint. The same platform (10 cm
diameter) was used for all phases of the water maze, and was
submerged 1.5 cm below the surface of the water. Before testing
began, mice were pre-trained for one day by placing them on the
platform in the middle of the pool. Mice typically jumped off the
platform the first 1–2 times. Once the mouse stayed on the
platform after being placed there, the next two trials consisted of
placing the mouse in the water at the edge of the pool and letting
the mouse swim to the platform. For the cued platform trials, a
black and white striped rod (2 cm diameter, 25 cm height) was
placed vertically in the center of the platform. The distance
traveled to reach the platform was measured in two sessions/day
for two days. Each session consisted of two trials in which mice
were dropped from different locations, and the platform was
located in different quadrants, to assess visual acuity and motor
performance. 60 s trials were separated by a 15 min inter-trial
interval.
Hidden platform training involved two sessions/day for five
days. Each session consisted of three trials. 60 s trials were
separated by a 15 min inter-trial interval. During acquisition trials,
the platform location remained constant and mice entered the
pool from randomized locations. 24 hours after the last training
trial, mice were given a 60 s probe trial test in which the platform
had been removed from the pool. Performance in the cued,
hidden, and probe trial portions of the maze was video-tracked
and analyzed using WaterMazeScan of the TopScan software
suite (CleverSys Inc, Reston, VA). No mice exhibited notable
floating or thigmotaxic behavior that affected performance. The
experimenter was blinded to genotypes during testing.
Statistical AnalysisImmunohistochemical data were analyzed using two-tailed,
unpaired Student’s t-tests. A p-value of less than 0.05 was
considered statistically significant. Behavioral data were analyzed
with two-way repeated measures ANOVA with Bonferroni post
hoc tests to compare the performance of hAPP mice relative to
NTG controls in the Morris water maze, as well in minute-by-
minute assessment for the tail suspension test. Probe trial
performance was assessed using one-way ANOVA followed by
Tukey post hoc tests for multiple comparisons. Two-way repeated
measures ANOVA was used to compare performances of hAPP
and NTG mice in the 24-hour trial of the tail suspension test
relative to performance in the baseline trial. One-way ANOVA
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with Newman-Kuels post hoc tests were used for multiple
comparisons tests on the same data.
Results
Hippocampal neurogenesis is impaired in hAPP mice at5–7 months of age
We examined doublecortin expression in the dentate gyrus of
the hippocampus in coronal sections from hAPP mice at 5–7
months of age, when behavioral/cognitive deficits are robust, but
before the development of extracellular plaques [23,25,26].
Doublecortin-expressing neuroblasts and immature neurons were
distinguished on the basis of morphology [20,21]. Neuroblasts are
only found in the subgranular zone, and appear as cell bodies
without any neuronal processes. Immature neurons can be found
in either the subgranular zone or the granule cell layer, and have
neuronal processes (Fig. 1A–B). We observed significant decreases
in numbers of neuroblasts in hAPP mice relative to NTG controls
(Fig. 1C, t[22] = 6.701, p,0.001). We also found significant
decreases in numbers of immature neurons in hAPP mice relative
to NTG controls (Fig. 1D, t[22] = 5.202, p,0.001).
Because the maturation of adult-born neurons is impaired in
hAPP mice [27], reductions in doublecortin-positive immature
neurons may reflect decreases in cell proliferation and/or the loss
of neuroblasts and immature neurons after cell proliferation has
occurred. To determine whether cell proliferation is reduced in
hAPP mice, we examined the incorporation of BrdU into cells in
the subgranular zone of the dentate gyrus of 5–7 month old hAPP
and NTG mice. We found that the numbers of BrdU-labeled cells
was reduced in hAPP mice relative to NTG controls (Fig. 1E–F;
t[11] = 3.076, p = 0.01), demonstrating that neurogenesis is
decreased in 5–7 month old hAPP mice.
hAPP mice do not exhibit depressive behavior at 5–7months of age
To determine whether impairments in adult neurogenesis
correspond with the appearance of depressive behavior in 5–7
month old hAPP mice, we assessed depressive behavior using the
tail suspension test. hAPP and NTG controls (n = 24/genotype)
were tested in a trial lasting six minutes. For each mouse, we
measured the latency to the first bout of immobility as well as the
total time spent immobile during the six minute trial. Reduced
latency to immobility and increased total time spent immobile
during the tail suspension test are indicative of a depressive
phenotype [22,28]. At 5–7 months of age, hAPP and NTG mice
exhibited similar latencies to the first bout of immobility (Fig. 2A).
hAPP and NTG mice also exhibited similar total durations spent
immobile during the six minute trial overall (Fig. 2B). Two-way
repeated measures ANOVA on the time spent immobile on a
minute by minute basis revealed an overall effect of genotype
(F[1,23] = 4.855; p,0.05) and time (F[1,115] = 31.11; p,0.001),
but none of the individual points were significant by Bonferroni
post-hoc tests (Fig. 2C). There was no interaction effect
(F[1,115] = 0.798; p = 0.55). Because 5–7 month old hAPP mice
did not display depressive behavior in baseline tail suspension test
trials, we examined whether a depressive phenotype might be
revealed with repeated tail suspension test trials. Repeated testing
in the tail suspension test induces a form of learned helplessness
that can be measured as an increased magnitude of depressive
behavior in subsequent trials [22,28]. Consistent with this
observation, NTG mice exhibited decreased latency to the first
bout of immobility when tested 24 hours after a baseline trial;
however, hAPP mice did not (Fig. 2D, n = 24/genotype). Two-way
repeated measures ANOVA revealed an interaction effect between
time and genotype, demonstrating that NTG mice reduced their
latency on the 24 hr trial relative to baseline, but hAPP mice did
not (F[1,46] = 13.46, p,0.001). One-way ANOVA followed by
Newman-Keuls multiple comparisons tests demonstrated that
there was an effect overall (F[3,92] = 13.40; p,0.001) and the
decrease in latency exhibited by NTG mice was significant
(p,0.001) but the slight decrease in hAPP mice was not (p.0.05).
Similarly, NTG mice exhibited increased total immobility time
over the six minute trial when tested 24 hours after a baseline trial,
whereas hAPP mice did not (Fig. 2E). Again, two-way repeated
measures ANOVA revealed an interaction between time and
genotype (F[1,46] = 11.90, p,0.005). One-way ANOVA followed
by Newman-Keuls multiple comparisons tests demonstrated that
there was an effect overall (F[3,92] = 7.214; p,0.001), and the
decrease in latency exhibited by NTG mice was significant
(q = 4.724, p,0.01) but there was no change in hAPP mice
(p.0.05). These results demonstrate that 5–7 month old hAPP
mice do not exhibit depressive behavior observable with the tail
suspension test either at baseline or with repeated testing.
We also examined the total immobility during only the first
three minutes of the tail suspension test, because the minute-by-
minute analysis in Fig. 2C indicated a separation between the
curves of NTG and hAPP mice during this time. When we
examined the total immobility over the first three minutes of the
test, we found that hAPP mice actually spent less time immobile
Figure 1. 5–7 month old hAPP mice exhibit impairments inneurogenesis. A, Micrographs of doublecortin immunostaining incoronal brain sections from NTG and hAPP mice. B, High magnificationmicrograph illustrating doublecortin-positive neuroblasts (Nb) andimmature neurons (ImN). C–D, Quantification of doublecortin expres-sion demonstrates significant decreases in neuroblasts (C) andimmature neurons (D) in hAPP mice relative to NTG controls. E–F,BrdU-labeling of dividing cells in the subgranular zone confirms adecrease in cell division in hAPP mice compared to NTG mice.Arrowheads, BrdU-labeled cells. n = 12/genotype. *p,0.05, ***p,0.001.doi:10.1371/journal.pone.0079651.g001
Neurogenesis and Depression in AD Mice
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over these first three minutes relative to NTG mice (Fig. 2F;
t[23] = 2.185, p,0.05).
We then examined whether this difference would affect the
measurement of learned helplessness in repeated testing. When we
re-analyzed the data using only the first three minutes of the tail
suspension test at both baseline and 24 hr trials, we confirmed that
NTG mice exhibited increased total immobility time when tested
24 hours after a baseline trial, whereas hAPP mice did not
(Fig. 2G). Two-way repeated measures ANOVA revealed effects of
both time and genotype (time: F[1,23] = 20.5, p,0.001; genotype:
F[1,23] = 15.76, p,0.001). There was no interaction effect
(F[1,23] = 2.909, p = 0.1). This result suggests that both NTG
and hAPP mice similarly altered their behavior at the 24 hr test
relative to baseline. However, one-way ANOVA followed by
Newman-Keuls multiple comparisons tests demonstrated that
there was an effect overall (F[3,46] = 12.00; p,0.001), and that
the increase in immobility exhibited by NTG mice at the 24 hour
test was significant (q = 4.874, p,0.01) but there was no significant
increase in immobility in hAPP mice at the 24 hr test (p.0.05).
Hippocampal-dependent memory is impaired in 5–7month old hAPP mice
Adult neurogenesis in the hippocampus is important for both
cognition as well as mood [9–11]. We and others have previously
demonstrated that several different lines of hAPP mice exhibit
hippocampal-dependent memory deficits [23–26]. To confirm
that 5–7 month old hAPP mice do exhibit memory impairments,
we tested mice in the Morris water maze. Mice were trained first in
the cued platform portion of the water maze, and then in the
hidden platform portion of the water maze, when the platform is
submerged beneath the surface of the water. In the acquisition
phase of the water maze (Fig. 3A), we found that hAPP mice were
impaired in the cued platform portion the Morris water maze
(F[1,22] = 6.676, p,0.05) although no individual points reached
statistical difference in Bonferroni post hoc tests; but hAPP and
NTG mice did not differ in the hidden platform acquisition phase
(F[1,22] = 3.973, p = 0.22). However, whereas NTG mice exhib-
ited a preference for the target quadrant relative to the other three
quadrants when tested in the probe trial, hAPP mice did not
(Fig. 3B–C, E; F[3,44] = 7.799, p,0.001 for NTG;
F[3,44] = 0.8519 for hAPP). In addition, the latency for hAPP
mice to reach the target area for the first time was higher than that
for NTG mice (Fig. 3D; t[22] = 2,652, p,0.05). There were no
differences in swim speed between NTG and hAPP mice, either in
the cued or hidden platform portions of the test, suggesting that
hAPP mice have no overt sensorimotor deficits (Fig. 3F; CUED
t[22] = 0.83, p = 0.415; HIDDEN t[22] = 0.61, p = 0.55). Swim
speeds were compared by assessing the average swim speeds over
all sessions in the cued or hidden platform portions of the test.
These results confirm that hAPP mice at this age have memory
impairments.
Impairments in hippocampal neurogenesis in hAPP miceat 13–15 months of age
We then examined doublecortin expression in coronal sections
from 13–15 month old hAPP mice to assess neurogenesis later in
disease progression, when both behavioral/cognitive deficits and
extracellular plaques are evident in hAPP mice [25,26]. At this age
NTG mice continue to demonstrate ongoing adult neurogenesis in
the hippocampus, although the number of neuroblasts and
immature neurons decreases with age, as expected (compare
Fig. 4A–D to Fig. 1A–D, see Table 1). For neuroblasts, there was
an average of 268.8612.02 in 5–7 month old mice vs 4362.6
neuroblasts in 13–15 month old NTG mice (t[22] = 18.35,
p,0.001). For immature neurons, there was an average of
232.2611.7 in 5–7 month old mice vs a 32.0862.4 immature
neurons in 13–15 month old NTG mice (t[22] = 16.67, p,0.001).
Neuroblasts and immature neurons maintain the same cell
morphology regardless of age (compare Fig. 4B to Fig. 1B).
Notably, relative to age-matched NTG controls, 13–15 month old
hAPP mice exhibit significant decreases in both neuroblasts
(t[21] = 7.527, p,0.001) and immature neurons (t[21] = 4.489,
p,0.001) in the dentate gyrus (Fig. 4C–D; n = 11–12/genotype).
We also found that the degree of neurogenesis impairments in
hAPP mice (relative to age-matched NTG controls) appeared to
increase with age. We calculated the level of neuroblasts and
immature neurons in hAPP mice as a percentage of age-matched
NTG controls in each age group, and then compared the relative
values of hAPP mice in the two age groups. The reduction in
Figure 2. 5–7 month old hAPP mice do not display depressivebehavior. A, Latency to the first bout of immobility in the tailsuspension test does not differ between hAPP and NTG mice. B, Thetotal time spent immobile during the 6-minute tail suspension test trialis not significantly different between NTG and hAPP mice. C, There is nosignificant difference between hAPP and NTG mice in the time spentimmobile during any individual minute of the trial. D, Repeated tailsuspension trials reveal that NTG mice exhibit a significant decrease inlatency to the first bout of immobility in the 24 hr trial relative to thebaseline trial, but there is no change in the latency of hAPP mice inrepeated trials. E, NTG mice spend more time immobile during a 24 hrtail suspension time trial relative to their baseline trial whereas hAPPmice do not change the time they spend immobile. F, Analyzing justthe first three minutes of the tail suspension test reveals that hAPP micespend less time immobile during the initial portion of the test. G,Analysis of the first three minutes of the tail suspension test at baselineand at the 24 hr trial confirms that NTG, but not hAPP, mice increasethe time spent immobile upon repeated testing. n = 12/genotype (A–C)and n = 24/genotype (D–E). **p,0.01, ***p,0.001.doi:10.1371/journal.pone.0079651.g002
Neurogenesis and Depression in AD Mice
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neuroblasts is greater in 13–15 month old hAPP mice compared to
5–7 month old hAPP mice (Table 1; t[21] = 2.5, p,0.05). The
reduction in immature neurons also appears to be greater in 13–15
month hAPP mice, although this did not reach statistical
significance (Table 1; t[21] = 1.085, p = 0.29).
To confirm that the reductions in doublecortin-expressing cells
in 13–15 month old hAPP mice are due to reduced cell
proliferation as exhibited by the 5–7 month old hAPP mice, we
examined the incorporation of BrdU into cells in the subgranular
zone of the dentate gyrus of 13–15 month old hAPP and NTG
mice. We found that the numbers of BrdU-labeled cells was
reduced in hAPP mice relative to NTG controls (Fig. 4E–F;
t[21] = 2.738, p,0.05).
hAPP mice exhibit a subtle depressive phenotype at 13–15 months of age
We tested 13–15 month old hAPP and NTG mice with the tail
suspension test to assess whether the exacerbated deficits in
neurogenesis evident in 13–15 month old hAPP mice with
advanced disease progression are associated with depressive
behavior. 13–15 month old hAPP mice demonstrated shorter
latencies to their first bouts of immobility relative to NTG controls
(Fig. 5A; t[21] = 2.559, p,0.05). Although the total time spent
immobile during the 6 minute trial did not differ between NTG
and hAPP mice (Fig. 5B; t[21] = 1.196, p = 2.449), analysis of the
time spent immobile on a minute-by-minute basis revealed that
hAPP mice became immobile sooner than did NTG mice (Fig. 5C).
Two-way repeated measures ANOVA revealed an interaction
between time and genotype (F[1,21] = 5.057, p,0.001), and
Bonferroni post hoc test demonstrated that hAPP mice spend
more time immobile already at the second minute of the six
minute trial (p,0.001).
To determine whether the depressive phenotype in hAPP mice
could be exacerbated by repeated testing in the tail suspension test,
we assessed hAPP and NTG mice in a second tail suspension test
24 hours after the baseline trial. At 13–15 months of age, both
NTG and hAPP mice mice demonstrated decreased latency to the
first bout of immobility on the 24 hour tail suspension test relative
to their baseline trials (Fig. 5D). One-way ANOVA followed by
Neuman-Keuls multiple comparisons tests demonstrated that the
Figure 3. Hippocampal-dependent memory deficits are ob-served in 5–7 month old hAPP mice. A, Performance in theacquisition portion of the Morris water maze. In the cued platformportion, 2-way repeated measures ANOVA indicates an overalldifference between NTG and hAPP mice (p,0.05), but no individualpoint reached significance in post-hoc tests. There was no significantdifference between NTG and hAPP mice in the hidden platform portionof the water maze. B–C, During the probe trial test for memory thatoccurred 24 hrs after the last training trial of the hidden platformacquisition phase, NTG mice spent more time in the target quandrantrelative to the other three quadrants (B) whereas hAPP mice did not (C).D, The latency for hAPP mice to reach the target area for the first timeduring the probe trial was higher than that for NTG mice. E,Representative paths swum during the probe trial for a NTG and anhAPP mouse. F, Swim speed was similar for NTG and hAPP mice duringboth the cued and hidden platform portions of the water maze. Swimspeeds were compared by assessing the average swim speeds over allsessions in the cued or hidden platform portions of the test. T, target;AR, adjacent right; AL, adjacent left; OP, opposite. n = 12/genotype.*p,0.05.doi:10.1371/journal.pone.0079651.g003
Figure 4. 13–15 month old hAPP mice exhibit decreasedneurogenesis. A, Micrographs of doublecortin immunostaining incoronal brain sections from NTG and hAPP mice. B, High magnificationmicrograph illustrating doublecortin-positive neuroblasts (Nb) andimmature neurons (ImN). C–D, Quantification of doublecortin expres-sion demonstrates significant decreases in neuroblasts (C) andimmature neurons (D) in hAPP mice relative to NTG controls. E–F,BrdU labeling of dividing cells in the subgranular zone demonstratesfewer dividing cells in the subgranular zone of hAPP mice. Arrowheads,BrdU-labeled cells. n = 11–12/genotype. *p,0.05, ***p,0.001.doi:10.1371/journal.pone.0079651.g004
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decrease in latency in both genotypes was significant
(F[3,42] = 19.32, p,0.001 overall; p,0.001 for NTG; p,0.05
for hAPP). However, two-way repeated measures ANOVA
revealed that time, but not genotype, induced a significant effect
on performance (F[1,21] = 50.91, p,0.001), which may reflect the
larger effect in NTG mice relative to hAPP mice. Indeed, there
was an interaction effect, demonstrating that time affected the two
genotypes differently (F[1,21] = 10.75, p,0.01). When we assessed
the total time spent immobile on the 24 hour trial relative to the
baseline trial, only NTG mice increased the time spent immobile
(Fig. 5E). Two-way repeated measures ANOVA showed that there
was a significant interaction effect between time and genotype
(F[1,21] = 21.75, p,0.001). One-way ANOVA followed by Neu-
man-Keuls multiple comparisons tests demonstrated that the
increase in immobility was significant for NTG but not for hAPP
mice (F[3,42] = 4.819, p,0.01 overall; p,0.01 for NTG; p.0.05
for hAPP). Together these results indicate that 13–15 month old
hAPP mice exhibit some, but not all, measures of increased
depressive behavior with repeated testing.
Because the minute-by-minute analysis in Fig. 5C indicated a
separation between the curves of NTG and hAPP mice during the
first three minutes of the tail suspension test, we also examined the
total immobility during only the first three minutes. We found that
hAPP mice spent more time immobile over these first three
minutes relative to NTG mice (Fig. 2F; t[21] = 2.66, p,0.05),
indicating that hAPP mice exhibit a subtle depressive phenotype at
13–15 months of age, an age at which neurogenesis is greatly
impaired.
Since hAPP mice did exhibit depressive behavior when
analyzing the initial portion of the tail suspension test, we then
examined whether analysing this portion of the test would reveal a
learned helplessness phenotype in hAPP mice with repeated
testing. When we re-analyzed the data using only the first three
minutes of the tail suspension test at both baseline and 24 hr trials,
we confirmed that NTG mice exhibited increased total immobility
time when tested 24 hours after a baseline trial. However, hAPP
mice did not (Fig. 2G). Two-way repeated measures ANOVA
showed that there was a significant interaction effect between time
and genotype (F[1,21] = 43.72, p,0.001). One-way ANOVA
followed by Neuman-Keuls multiple comparisons tests demon-
strated that the increase in immobility was significant for NTG but
not for hAPP mice (F[3,42] = 13.57, p,0.001 overall; p,0.001 for
NTG; p.0.05 for hAPP). Together these results indicate that
although 13–15 month old hAPP mice exhibit depressive
behaviors, they do not exhibit a learned helpessness phenotype
with repeated testing.
Hippocampal-dependent memory is impaired in 13–15month old hAPP mice
We also tested 13–15 month old NTG and hAPP mice in the
Morris water maze to assess hippocampal-dependent spatial
memory. We found that hAPP mice were impaired in the cued
portion of the water maze (Fig. 6A), although they did exhibit
learning throughout the acquisition sessions (F[1,16] = 24.61,
p,0.001). hAPP mice were also significantly impaired in
acquisition of the hidden platform portion of the water maze
(F[1,16] = 9.653, p,0.01). Notably, NTG mice performed well in
the probe trial (Fig. 6B, E), spending more time in the target
quadrant relative to the other three quadrants (F[3,40] = 6.291,
p,0.005; Tukey post-hoc tests p,0.05). However, hAPP mice
exhibited memory impairment in the probe trial (Fig. 6C, E).
Although two-way repeated measures ANOVA indicated an
overall difference (F[3,24] = 3.532, p,0.05), Tukey post-hoc tests
revealed that hAPP mice only exhibited a preference for the target
quadrant relative to the adjacent left quadrant, but not to the other
two quadrants (p,0.05 vs adjacent left; p.0.05 vs adjacent right
and opposite quadrants). We also found that the latency for hAPP
mice to reach the correct quadrant area for the first time was
higher than that for NTG mice (Fig. 6D; t[16] = 2.43, p,0.05).
These results indicate that hippocampal-dependent memory
impairments persist with age in hAPP mice. We found that
although the swim speed of hAPP mice was slower than that of
NTG mice in the cued portion of the water maze (Fig. 6F;
t[1,16] = 2.62, p,0.05), there was no difference in swim speed
between NTG and hAPP mice in the hidden platform portion of
the test (Fig. 6E; t[1,16] = 1.45, p = 0.167). Because swim speed
was similar in the hidden platform portion of the test, this indicates
that there is likely no overt sensorimotor deficits in hAPP mice, but
suggests that other factors may have affected performance in the
cued portion of the test.
Discussion
In this report, we demonstrate that hAPP mice exhibit
significant impairments in neurogenesis by 5–7 months of age
that worsen with age, but subtle depressive behavior in the tail
suspension test is only measurable by 13–15 months of age.
However, hAPP mice exhibit impairments in spatial memory
already by 5–7 months of age. We and others have observed
Table 1. Age-related changes in doublecortin-expressing cells.
5–7 mo old 13–15 mo old p-value
Neuroblasts:
NTG vs NTG (number of cells) 268.8612.02 4362.6 t[22] = 18.35, p,0.001
hAPP vs hAPP (number of cells) 134616.06 10.9163.42 t[22] = 7.209, p,0.001
hAPP vs hAPP (rel. to age-matched NTG) 0.560.06 0.2560.08 t[21] = 2.50, p,0.05
Immature Neurons:
NTG vs NTG (number of cells) 232.2611.7 32.0862.4 t[22] = 16.67, p,0.001
hAPP vs hAPP (number of cells) 112.5619.77 1064.44 t[21] = 4.854, p,0.001
hAPP vs hAPP (rel. to age-matched NTG) 0.48560.085 0.31260.138 t[21] = 1.085, p = 0.29
Numbers of neuroblasts and immature neurons in each age group and genotype are listed for comparative purposes. Three comparisons are provided: 1) numbers ofcells in 5–7 mo old NTG mice vs 13–15 mo old NTG mice; 2) numbers of cells in 5–7 mo old hAPP mice vs 13–15 mo old hAPP mice; and 3) the proportion of cells in 5–7 mo old hAPP mice relative to age-matched NTG mice vs the propotion of cells in 13–15 mo old hAPP mice relative to age-matched NTG mice.doi:10.1371/journal.pone.0079651.t001
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PLOS ONE | www.plosone.org 6 November 2013 | Volume 8 | Issue 11 | e79651
memory deficits in several versions of the water maze in this line of
mice [23,29–32], but a comparison to the development of
depressive behaviors has not previously been described.
The fact that no depressive behavior is evident at 5–7 months,
when hAPP mice already display significant impairments in
neurogenesis, suggests that these phenotypes may not be tightly
linked in hAPP mice. Indeed, hAPP mice exhibited less immobility
in the first half of the tail suspension test, consistent with a less
depressive phenotype. Even with repeated testing in the tail
suspension test, hAPP mice did not show a decrease in latency to
immobility nor an increase in total immobility as did NTG mice,
which is again consistent with a less depressive phenotype. Older
hAPP mice at 13–15 months of age did exhibit depressive
behaviors. However, because the depressive behavior exhibited by
13–15 month old hAPP mice is associated with greater deficits in
neurogenesis than are evident at 5–7 months, it is possible that
more advanced impairments in neurogenesis are necessary before
the development of a depressive phenotype. Notably, although 13–
15 month old hAPP mice exhibited a depressive phenotype, they
did not exhibit learned helpessness with repeated testing in the tail
suspension test, which may be a indication of a failure to adapt
behavior as a result of experience. Indeed, it is noteworthy that
whether hAPP mice spend less time immobile during the first
3 minutes of the trial (as for the 5–7 month old mice, Fig. 2F) or
whether they spend more time immobile during the first 3 minutes
of the trial (as for the 13–15 month old mice, Fig. 5F), hAPP mice
do not exhibit any differences between the first and second tail
suspension tests, whereas NTG mice do (Fig. 2G and 5G).
Figure 5. 13–15 month old hAPP mice display subtle depres-sive behavior. A, In the tail suspension test, hAPP mice exhibitdecreased latency to the first bout of immobility relative to NTGcontrols. B, No significant difference between hAPP and NTG mice inthe total time spent immobile during the 6-minute tail suspension testtrial. C, hAPP mice become immobile sooner than do NTG mice duringthe tail suspension trial. D, Both NTG and hAPP mice demonstratedecreased latency to the first bout of immobility in response to arepeated tail suspension test. E, NTG mice increase the time spentimmobile during a repeated tail suspension test, whereas there is nochange in hAPP mice. F, Analyzing just the first three minutes of the tailsuspension test reveals that hAPP mice spend more time immobileduring the initial portion of the test. G, Analysis of the first threeminutes of the tail suspension test at baseline and at the 24 hr trialconfirms that NTG, but not hAPP, mice increase the time spentimmobile upon repeated testing. n = 11–12/genotype. *p,0.05, **p,0.01, ***p,0.001.doi:10.1371/journal.pone.0079651.g005
Figure 6. Hippocampal-dependent memory deficits are ob-served in 13–15 month old hAPP mice. A, Performance in theacquisition portion of the Morris water maze. In the cued platformportion, 2-way repeated measures ANOVA indicates an overalldifference between NTG and hAPP mice (p,0.01). Asterisk denotesp,0.05 for individual data points. There was also a significant differencebetween NTG and hAPP mice in the hidden platform portion of thewater maze, although no individual point reached significance in post-hoc tests (2-way repeated measures ANOVA p,0.01). B–C, During theprobe trial test for memory that occurred 24 hrs after the last trainingtrial of the hidden platform acquisition phase, NTG mice spent moretime in the target quandrant relative to the other three quadrants (B)whereas hAPP mice did not (C). D, The latency for hAPP mice to reachthe correct quadrant for the first time during the probe trial was higherthan that for NTG mice. E, Representative paths swum during the probetrial for a NTG and an hAPP mouse. F, Swim speed was lower for hAPPmice than NTG mice in the cued portion of the water maze, but wassimilar to NTG mice during the hidden platform portion of the maze.Swim speeds were compared by assessing the average swim speedsover all sessions in the cued or hidden platform portions of the test.n = 7–11/genotype. T, target; AR, adjacent right; AL, adjacent left; OP,opposite. *p,0.05, **p,0.01.doi:10.1371/journal.pone.0079651.g006
Neurogenesis and Depression in AD Mice
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If advanced deficits in neurogenesis in hAPP mice are required
for the development of a depressive phenotype, then how might
these impairments be related to depression in AD, which often
manifests early in disease progression [33,34]? Although deficits in
neurogenesis are associated with depression, reduced neurogenesis
alone is not sufficient to induce depression in rodent models
[16,17]. However, early deficits in neurogenesis in AD may allow
other pathological symptoms of the disease to cause depression. If
reduced neurogenesis acts as a permissive factor for the
development of depression, and impairments in neurogenesis
worsen with age and disease progression, then more advanced
deficits in neurogenesis may be present by the time depression
manifests in AD. Consistent with this possibility, the therapeutic
effects of several classes of antidepressants rely on mechanisms that
increase neurogenesis [16–18].
It is important to also consider the possibility that the tail
suspension test may not be sensitive enough to detect a subtle
depressive phenotype that might exist in early stages of disease in
hAPP mice. The tail suspension test is widely used as a robust
assay for assessing the therapeutic effects of antidepressants [28];
however, a more ethologically-relevant behavioral task may be
better able to detect subtle mood disorders in our mice. To address
this possibility, we also tested hAPP and NTG mice with the forced
swim test [22,28], as this test has been previously used to assess
depressive behavior in an AD mouse model [35]. In our study,
however, we found that results were complicated by anxiety-
related behaviors exhibited by hAPP mice when placed in the task
chamber (unpublished observations), making it difficult to
interpret the data. hAPP mice did not exhibit any anxiety-related
behaviors in the tail suspension test. A different type of
ethologically-relevant behavioral paradigm such as the resident
intruder test [36] may better assess mild depressive behavior in
hAPP mice and reveal a depressive phenotype at a younger age.
Regardless, our results indicate that although robust impairments
in neurogenesis are observed early in disease progression in hAPP
mice, a depressive phenotype is not overt, and reaches only subtle
indications at older ages. Together these results suggest that
depressive behaviors and impairments in neurogenesis and are not
tightly linked in hAPP mice.
Whereas subtle depressive behavior is only evident in hAPP
mice by 13–15 months of age, the inability of these mice to learn
and modify their behavior in response to repeated tail suspension
trials is apparent by 5–7 months of age. At this age (5–7 mo),
hAPP mice also already exhibit spatial memory deficits in the
water maze. Together, these observations underscore the fact that
whereas depression is a common symptom of AD, the most
prominent symptom of AD is cognitive deficit, particularly related
to learning and memory [1,33,37].
Author Contributions
Conceived and designed the experiments: DMI JC. Performed the
experiments: DMI SP MSP XZ LZ. Analyzed the data: DMI SP MSP
XZ LZ JC. Wrote the paper: DMI JC.
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